The following description provides a summary of information relevant to the present disclosure and is not a concession that any of the information provided or publications referenced herein is prior art to the presently claimed current invention.
The SELEX process is a method for the in vitro evolution of nucleic acid molecules that are able to bind with high specificity to target molecules and is described in U.S. Pat. No. 5,475,096 entitled “Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163 (see also WO 91/19813) entitled “Nucleic Acid Ligands” each of which is specifically incorporated by reference herein in its entirety. These patents, collectively referred to herein as the SELEX Patents, describe methods for making a nucleic acid ligand to any desired target molecule.
The basic SELEX process has been modified to achieve a number of specific objectives. For example, U.S. Pat. No. 5,707,796, entitled “Method for Selecting Nucleic Acids on the Basis of Structure” describes the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938, entitled “Nucleic Acid Ligands to HIV-RT and HIV-1 Rev” describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. Pat. No. 5,705,337, entitled “Systematic Evolution of Ligands by Exponential Enrichment Chemi-SELEX” describes methods for covalently linking a nucleic acid ligand to its target.
The SELEX process encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, see supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′—NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe).
Further modifications of the SELEX process are described in U.S. Pat. No. 5,763,177, U.S. Pat. No. 6,001,577, and U.S. Pat. No. 6,291,184, each of which is entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX”; see also, e.g., U.S. Pat. No. 6,458,539, entitled “Photoselection of Nucleic Acid Ligands”. These patents, collectively referred to herein as “the PhotoSELEX Patents,” describe various SELEX methods for selecting nucleic acid ligands containing photoreactive functional groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. The resulting photoreactive nucleic acid ligands are referred to as photocrosslinking nucleic acid ligands or photoaptamers.
Although these SELEX and photoSELEX processes are useful, there is always a need for processes that lead to improved properties of nucleic acid ligands generated from in vitro selection techniques. For example, a need exists for a method for producing nucleic acid ligands to target molecules with better binding affinities than those achieved with naturally occurring DNA or RNA nucleotides. Additionally, a need exists for a method for producing photoaptamers with increased photocrosslinking yields and enhanced photo-selectivity. Also, there is a need for methods for producing nucleic acid ligands, both affinity- and photo-aptamers, with a minimal number of nucleotides required to impart the properties of interest.